Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation.

Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.

A high-resolution spatiotemporal atlas of gene expression of the developing mouse brain.

To provide a temporal framework for the genoarchitecture of brain development, we generated in situ hybridization data for embryonic and postnatal mouse brain at seven developmental stages for ∼2,100 genes, which were processed with an automated informatics pipeline and manually annotated. This resource comprises 434,946 images, seven reference atlases, an ontogenetic ontology, and tools to explore coexpression of genes across neurodevelopment. Gene sets coinciding with developmental phenomena were identified. A temporal shift in the principles governing the molecular organization of the brain was detected, with transient neuromeric, plate-based organization of the brain present at E11.5 and E13.5. Finally, these data provided a transcription factor code that discriminates brain structures and identifies the developmental age of a tissue, providing a foundation for eventual genetic manipulation or tracking of specific brain structures over development. The resource is available as the Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org).

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.

Cell type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

Divergent and nonuniform gene expression patterns in mouse brain.

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs.

Genome-wide atlas of gene expression in the adult mouse brain.

Molecular approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behaviour. The cellular diversity of the brain necessitates a cellular resolution approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas containing the expression patterns of approximately 20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural analysis and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resolution analysis has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.

Synthesis and antibacterial activity of novel C12 ethyl ketolides.

A novel series of C(12) ethyl erythromycin derivatives have been discovered which exhibit in vitro and in vivo potency against key respiratory pathogens, including those resistant to erythromycin. The C(12) modification involves replacing the natural C(12) methyl group in the erythromycin core with an ethyl group via chemical synthesis. From the C(12) ethyl macrolide core, a series of C(12) ethyl ketolides were prepared and tested for antibacterial activity against a panel of relevant clinical isolates. Several compounds were found to be potent against macrolide-sensitive and -resistant bacteria, whether resistance was due to ribosome methylation (erm) or efflux (mef). In particular, the C(12) ethyl ketolides 4k,4s,4q,4m, and 4t showed a similar antimicrobial spectrum and comparable activity to the commercial ketolide telithromycin. The in vivo efficacy of several C(12) ethyl ketolides was demonstrated in a mouse infection model with Streptococcus pneumoniae as pathogen.

Synthesis and antibacterial activity of novel C12 vinyl ketolides.

A novel series of C12 vinyl erythromycin derivatives have been discovered which exhibit in vitro and in vivo potency against key respiratory pathogens. The C12 modification involves replacing the natural C12 methyl group in the erythromycin core with a vinyl group via chemical synthesis. From the C12 vinyl macrolide core, a series of C12 vinyl ketolides was prepared. Several compounds were found to be potent against macrolide-sensitive and -resistant bacteria. The C12 vinyl ketolides 6j and 6k showed a similar antimicrobial spectrum and comparable activity to the commercial ketolide telithromycin. However, the pharmacokinetic profiles of C12 vinyl ketolides 6j and 6k in rats differ from that of telithromycin by having higher lung-to-plasma ratios, larger volumes of distribution, and longer half-lives. These pharmacokinetic differences have a pharmacodynamic effect as both 6j and 6k exhibited better in vivo efficacy than telithromycin in rat lung infection models against Streptococcus pneumoniae and Haemophilus influenzae.